Strain of measles virus and process of producing the same

ABSTRACT

The disclosure describes a process for the production of an attenuated strain of measles virus effective for the production of an attenuated measles virus vaccine. The starting material is an Edmonston strain of measles virus which has undergone a plurality of culture passages in various culture media. A sample of the last culture passage is taken and a plurality of culture passages of the sample is effected in primary cultures of fibroblasts of chicken embryos. A sample from the last culture passage is inoculated and a culture passage is effected in a primary culture of fibroblasts of chicken embryos thereby obtaining clones of virus. Clones of measles virus obtained in the last culture passage are isolated and one selects those which are thermosensitive at a temperature between about 32° C. and 37° C. Among the thermosensitive clones which have been selected, another selection is made of the ones with moderate cytopathic effect, and finally one isolates from the latest selection, the one which shows highest seroconversion on monkeys, thus producing an attenuated strain of measles virus.

This invention relates to a process for the production of an attenuatedstrain of measles virus and to the new strain of measles virus thusobtained. More particularly, the invention relates to an attenuatedmeasles virus vaccine and to a process for the preparation thereof.

Measles is a disease caused by a virus which is suffered both bychildren and adults. This disease is well under control in the highlydeveloped countries because of the possibilities of vaccinations or goodmedical care of the persons which have not been vaccinated and who mighthave caught the disease. However, in under-developed countries, thisdisease reaches nearly catastrophic levels and therefore it is highlydesirable that any efforts be made to provide suitable relief either byvaccination or other kinds of treatments.

It is well known that an individual which has not been immunized can beprotected against the attack of a disease of viral nature be beingimmunized with a vaccine containing a living virus of the same nature,which is immunologically related to the virulent virus but which is notitself pathogenic. Such a vaccine may contain an attenuated strain whichis derived by various known treatments from the virulent virus. Avaccine is suitable for vaccination if the virulence of the virus thatit contains is low. The term virulence signals the degree of damage agerm (microbe) is able to cause to the organism it has invaded, undernormal circumstances.

Therefore, like any other disease of viral origin, measles can andshould be treated by vaccination with an attenuated measles virus. It iswell known that in some other diseases of infectious nature, a dead germcan perform the function of the vaccine. However, such is not the casewith measles and it is known that a measles vaccine should contain aliving measles virus which is of course attenuated. The idea is to comeup with a vaccine which has as little undesired secondary effects aspossible.

The prior art is loaded with examples of vaccines which are produced byfirst obtaining attenuated forms of measles virus and incorporatng themicro-organism in some kind of diluent for injection to a person.

For example, the measles virus in virulent form was previously culturedin tissue culture on various substrates and it was allowed to multiplyin chick embryo tissue. One of the most popular forms of measles virusvaccine has been derived from the Edmonston strain which has beendeveloped by Enders et al (Enders, Katz, Medearis "Recent Advances inKnowledge of Measle Virus" in Perspectives in Virology, New York, JohnWiley, 1959; Katz, Milovanovic and Enders, Proceedings of the Societyfor Experimental Biology and Medicine, vol. 97, pp. 23-29, 1958). TheEdmonston strain was produced by Enders as a result of successive seriesof passages through human kidney tissue culture, human amnion tissueculture, embryonated eggs and chick embryo tissue culture. According tothese methods of culture the virus was incubated in the culture medium,after incubation at temperatures between 35° C. and 37° C. The resultingcultured virus is attenuated to some extent which means that it ispartially modified. However, it has been found out that inoculation ofnonimmune humans produces relatively severe undesirable side effects,such as high fevers and the like. Also, children which have beenimmunized with the vaccine based on the Edmonston strain of measlesvirus often suffered from rash which is of course not acceptable. Itwill therefore be seen that the Edmonston strain was not completelyacceptable because of the side effects especially in children. It wasfound that immunization with the Edmonston strain had to be accompaniedby an injection of gamma globulin to protect the child from severesymptoms. This is of course not practical and it is quite expensive.

Schwartz in U.S. Pat. No. 3,133,861 describes a process for producing anattenuated measles virus vaccine which allegedly produces little or noundesirable reaction in children which are inoculated with the vaccine.The method broadly consists in culturing a live measles virus in avianembryo tissues at a temperature of about 28° C. to about 32° C. using arepeated number of culture passages until the attenuated virus producesin humans no or little undesirable reaction to the vaccine. The numberof passages could be quite large, for example at least about 40 andtherefore the process is quite complex, let alone time consuming.

We have found that it is possible to produce an attenuated strain ofmeasles virus which is effective for the production of measles virusvaccine and which is free or substantially free of side effects, by amethod which comprises:

selecting an Edmonston strain of measles virus which has undergone aplurality of culture passages in various culture media and taking asample of the last culture passage;

effecting a plurality of culture passages of the sample obtained, inprimary cultures of fibroblasts of chicken embryos;

selecting a sample from the last culture passage and effecting at leastone culture passage thereof in a primary culture of fibroblasts ofchicken embryos;

isolating clones of measles virus obtained in the last culture passageand selecting those which are thermosensitive at a temperature betweenabout 32° C. and 37° C.;

among the thermosensitive clones which have been selected, makinganother selection of the ones with moderate cytophathic effect; and

in final instance, isolating from the latest selection, the one whichshows highest seroconversion on monkeys, thus producing an attenuatedstrain of measles virus.

Although the number of passages which the measles virus has to undergothrough the various culture medium can vary to a large extent, the ideais that, in final analysis, the strain must be sufficiently attenuatedto prevent the formation of side effects in the person which is beingimmunized. The Edmonston strain which has been subjected to thefollowing culture passages:

(a) Twenty-four (24) culture passages in human kidney cells;

(b) Twenty-eight (28) culture passages in human amniotic cells;

(c) Six (6) culture passages in the amniotic sac of embryonated eggs;

(d) Thirteen (13) culture passages in cells of chicken embryos; followedby

(e) Eleven (11) culture passages in cells of chicken embryos was used byus for this purpose.

In accordance with the preferred embodiment of the invention, the virussample is thereafter subjected to a first culture passage in a primaryculture of fibroblasts of chicken embryos maintained at 33° C. followedby a second culture at 33° C. passage in a primary culture offibroblasts of chicken embryos.

The clones of measles virus from the second culture passage in a primaryculture of fibroblasts of chicken embryos are isolated by inoculatingthe strain in Petri dishes kept in an incubator in a 5% CO₂ enrichedatmosphere during about two hours at about 32° C., after which thecultures are drained and washed away using phosphate buffer saline, anda culture medium is added and incubation is allowed to take place forfour to six days at 32° C., after which the liquid medium is replaced bya methyl cellulose medium and the cultures are again incubated at 32° C.for a period of about 3 to 4 days, until plaques appear and differentclones can be separated. This is a well known technique which, accordingto the invention, has been used for the first time in furtherattenuating this virus strain.

The separation of the clones and the selection of the one which is mostsuitable for producing measles vaccine can be carried out in a number ofways. However, we found that it would be preferable to selectfourty-seven (47) samples of virus from the clones which have beenseparated, which are incubated on chicken embryo primary cultures for anincubation period for about fourteen (14) days, after which seven (7)are retained which are thermosensitive at a temperature between about32° C. and 37° C. Each of the seven (7) samples selected is treated forthe purpose of isolating clones by producing plaques by the technicalmethod described above. Each plaque is again isolated in order to selectthree (3) clones with moderate cytopatic effect, and finally, one of thethree clones selected, the one which has highest seroconversion onmonkeys, is separated and is frozen at a temperature of -70° C.

In order to produce a vaccine which is stable for a certain period oftime, various stabilizers could be added. However, it has been foundout, according to the invention, that especially for the purpose ofusing a vaccine in tropical climate, it would be useful to use astabilizer which consists of dextran, monosodium glutamate, d-sorbitoland normal serum albumin (human).

Preferably, the attenuated measles virus is suspended in the harvestmedium containing the live virus and the final concentration of theingredients of the stabilizer in the virus suspension are the following:

    ______________________________________                                            1. Dextran (NBC) MW 60 000 to 90 000                                                                    5.0%                                                2. Sodium glutamate (mono), (Fisher N° S-302                                                     1.0%                                                3. d-Sorbitol (hydrate) (glucitol)                                        (General Biochemicals N° 50180)                                                                      1.0%                                                4. Normal Serum Albumin (Human) (25% solution)                            (Hyland N° 060-016)    0.5%                                            ______________________________________                                    

Other formulas, equally efficient are as follows:

    ______________________________________                                        (2)    Dextran                 5.0%                                                  Monosodium glutamate    1.0%                                                  d-Sorbitol              0.5%                                                  Normal Serum Albumin in 25% solution                                                                  1.0%                                           (3)    Dextran                 5.0%                                                  Monosodium glutamate    1.0%                                                  d-Sorbitol              1.0%                                                  Normal Serum Albumin in 25% solution                                                                  1.0%                                           (4)    Dextran                 6.0%                                                  Monosodium glutamate    1.0%                                                  d-Sorbitol              1.0%                                                  Normal Serum Albumin in 25% solution                                                                  0.5%                                           (5)    Dextran                 6.0%                                                  Monosodium glutamate    1.0%                                                  d-Sorbitol              0.5%                                                  Normal Serum Albumin in 25% solution                                                                  1.0%                                           (6)    Dextran                 6.0%                                                  Monosodium glutamate    1.0%                                                  d-Sorbitol              1.0%                                                  Normal Serum Albumin in 25% solution                                                                  1.0%                                           ______________________________________                                    

We will now give, for illustration purposes only an example ofproduction of the new attenuated strain.

Source of virus:

An Edmonston strain of measles virus was obtained from Doctor J. F.Enders by Lederle Laboratories of American Cyanamid Company, PearlRiver, N.Y. after the measles virus has been subjected to the followingculture passages:

1. Twenty-four (24) culture passages in human kidney cells;

2. Twenty-eight (28) culture passages in human amniotic cells;

3. Six (6) culture passages in the amniotic sac of embryonated eggs;

4. Thirteen (13) culture passages in chick embryo cells.

After receiving this strain, Lederle Laboratories has subjected thevirus to eleven (11) culture passages on chick embryo cells. A sample ofthe eleventh passage, in the form of a lyophilized vaccine was used asthe starting material.

The virus was cultured a first time on a primary culture of fibroblastsof chicken embryos maintained at 33° C. The virus which has beenlarvested was distributed into many flasks and was kept at -75° C. Thesource designated "S-37" which was used for the production of batches ofvirus vaccine has been obtained from this first passage by means of asecond culture passage on a primary culture of fibroblasts of chickenembryos. The absence of adventitious virus in the cultures used forthese two passages was controlled by subcultures on cultures offibroblasts of chicken embryos, on kidney cells of Cercopithecitae, onkidney cells of Rhesus monkey and by the RIF and Cofal tests.

Appropriate dilutions of the source S-37 have been inoculated on primarycultures of fibroblasts of chicken embryos in Petri dishes kept in anincubator in a 5% Co₂ enriched atmosphere during about two hours at 32°C. The cultures are then drained and washed with phosphate buffer salineafter which more culture medium is added and incubation is allowed totake place for four to six days at 32° C. The liquid medium is thenreplaced by adding methyl cellulose and the cultures are then allowedagain to stand at 32° C. for a period of three to four days. Thecellular layer is then colored with neutral red at a concentration of1:30000. After a careful microscopic examination, fourty-seven (47)plaques containing clones of virus were isolated. The virus of each ofthese plaques is cultured into Petri dishes containing fibroblasts ofchicken embryo in liquid medium: one of the dishes was incubated at 37°C. and the other one at 32° C. The reason for this is that it isintended to determine which of the particular virus which is isolatedstill grows at 32° C. but stops growing at 37° C. In other words, thestrain which is intended to be obtained should have little or nocytopatic effect at 37° C. After fourteen days of incubation, tests weremade to determine which of the virus is still growing at 32° C. but isnot growing at 37° C. In other words, these tests are made in order todetermine those which are thermosensitive between 32° C. and 37° C. Thevalue of 32° C. has been selected as the most practical. Of course, onecould select a slightly lower or higher values although it has beenfound out that 32° C. is the most easy temperature with which operate.It was found out that seven (7) clones of virus have substantially nocytopatic effect at 37° C., while for these same virus, this effect wasstill there in cultures maintained at 32° C.

The liquids which are at the surface of the cultures maintained at 32°C. of these seven (7) sources were used to produce plaques by the sametechnical procedure is described above. Five (5) plaques werere-isolated from each of these sources and were separately tested fortheir cytopatic effects in liquid medium at 32° C. and 37° C. Tests werethereafter made to determine which of the sources are most suitable toprovide a new strain, i.e. those which have the best morphology of theplaques and have the least cytopatic effect. Three (3) sources wereretained which are designated as IMH-1, IMH-2 and IMH-3. Various testswere made with these three (3) sources and the IMH-2 was retained as asource of vaccine since, on the one hand, it gives products of betterquality and on the other hand, the serologic conversion on monkey afterintracerebral inoculation is also higher.

In the photos which illustrate the invention:

FIG. 1 is a microscopic enlargement of plaques of the Edmonston strainof measles virus;

FIG. 2 is a microscopic enlargement of plaques of the Schwarz strain;

FIG. 3 is a microscopic enlargement of the IMH-2 strain; and

FIG. 4 is a microscopic enlargement of a strain called "L'OISEAU" of ameasles virus isolated from a clinical case of measles.

From the photos, it will be seen that the strain IMH-2 seems the bestsuitable for reproducing a strain of attenuated measles virus.

The source IMH-2 was selected as a result of various tests as indicatedabove, the main one being the serologic conversion on monkeys. It wasfound out that with the IMH-2 there was 90% seroconversion while withthe IMH-1 it was only 75% seroconversion. The strain selected wastherefore the IMH-2 which was used to produce a measles virus vaccine.

Once a suitable attenuated strain of measles virus is obtained it wasdecided to add a stabilizer to the vaccine having in mind that it willbe used in countries with tropical climate coupled with long distancetransportation. The question of temperature is very important.

Under these circumstances, a good stabilizer should offer the bestprotection possible during freeze-drying, it should provide good storageof the vaccine at 4° C. (refrigerator temperature) as well as at -25° C.

Obviously, after freeze-drying, the vaccine should remain quite intact.In view of the fact that good protection should be afforded at -25° C.as well as at 4° C. for as long as possible, it was found that a goodstabilizer should contain the following ingredients:

    ______________________________________                                        1. Dextran;                                                                   2. sodium monoglutamate;                                                      3. d-sorbitol;                                                                4. normal serum albumin (human)                                               As an example, the following formula was used.                                Virus, suspended in the harvest Medium 199 (Earle)                                                       = 75%                                               concentrated stabilizer 9/4                                                                             = 25%                                              ______________________________________                                    

In the suspension obtained, the final concentration of the ingredientspertaining to the stabilizer are:

    ______________________________________                                        1.   Dextran (NBC) MW 60 000 to 90 000                                                                          5.0%                                        2.   Sodium glutamate (mono), (Fisher N° S-302)                                                          1.0%                                        3.   d-Sorbitol (hydrate) (glucitol)                                               (General Biochemicals N° 50180)                                                                     1.0%                                        4.   Normal Serum Albumin (Human) (25% solution)                                   (Hyland N° 060-016)   0.5%                                        ______________________________________                                    

One vial of 20 human doses contains 2.7 ml of the freeze-driedsuspension, representing about 0.2 g of solid matter. To reconstitutethe vaccine, 10 ml of diluent has to be added, bringing the final volumeup to about 10.2 ml, in which each 0.5 ml represents one human dose.

The dilution factor is 10.2:2.7=3.7778. Thus, in the reconstitutedvaccine the proportions are:

    ______________________________________                                        Dextran, 5:3.7778       = 1.3235%                                             Monosodium glutamate, 1:3.7778                                                                        = 0.2647%                                             d-Sorbitol, 1:3.7778    = 0.2647%                                             Human serum albumin (25%), 0.5:3.7778                                                                 = 0.13235%                                            ______________________________________                                    

The specific weight of the virus suspension with the stabilizerincorporated being about 1.026, the weight correction per human dose of0.5 ml is 0.5×1.026=0.513 g and each human dose of 0.5 ml contains:

    __________________________________________________________________________      Dextran, 1.3235%; (0/513 × 1.3235): 100 = 6.7896.sup.-3 g                                            =6.7896mg                                        Monos.glutam., 0.2647%; (0.513 × 0.2647): 100 = 1.3579.sup.-3                                        = 1.3579mg                                       d-Sorbitol, 0.2647%          = 1.3579mg                                       Hum.serum albumin (25%), 0.13235%; (0.513 × 0.13235):                   100 = 0.6789.sup.-3 g        = 0.6789mg                                     __________________________________________________________________________

Different tests were made to determine the value of the new vaccine.

In a first test, studies were made to determine the seroconversion andto find the presence of virus in the throat.

Four (4) children, aged 9 to 12 months, were treated. At that time,tthey had no antibodies capable of neutralizing the measles virus.

Daily clinical examination

Local reaction: little blotch and induration the 3th day aftervaccination, in one case (R-02);

Rectal temperature: 39.6° C. on the 9th day, followed by a small rash onthe 11th and the 12th day on one child (R-04).

Nothing else was observed on these children.

The neutralizing antibodies 100 TCID₅₀ in tubes of BSC-1 cells at 33° C.were

    ______________________________________                                        before vaccination                                                                             after vaccination                                            ______________________________________                                        R-01 < 1/4       1/28                                                         R-02 < 1/4       1/18                                                         R-03 < 1/4       1/76                                                         R-04 < 1/4       1/58                                                         ______________________________________                                    

Swabbing out of throat

The samples were each cultured in 4 tubes of kidney cultures ofcercopithecus. They were observed during 13 days and no cytopathiceffect was attributable to the measles virus. The floating liquids werecollected 13 days after the first culture. They were cultured again 13days later during which they were carefully observed on each day.Negative results.

Another test was made to study the seroconversion and to find out thepossible transmission of the virus.

Nineteen (19) children aged between 15 months and 10 years and 8 monthsold were observed on a dayly basis during 46 days. None of thesechildren had any antibodies capable of neutralizing the measles virus atthe time of the vaccination.

Twelve (12) children were vaccinated while the 7 others have received adiluent and these children were used as witnesses to study the possibletransmission of the virus.

The children were kept 3 in each room, i.e. 2 who have been vaccinatedand one witness, during 15 days.

Daily clinical examination

    ______________________________________                                        Local reaction:                                                                            None.                                                            Rectal temperature:                                                                        on those vaccinated:                                                        no. 326-1                                                                             -38.9°  C. the 5th                                                      and 12th days                                                        no. 305 -39.4° C. the 9th day                                                   38.9°  C. the 10th and                                                 11th days                                                            on the witnesses:                                                             no. 319 -39.4°  C. the 4th day                                                  with coryza                                                          no. 305 -38.9°  C. the 5th day                                                  with coryza                                                          no. 326-3                                                                             -38.9°  C. the 12th, 13th                                               26th, 27th and 33th                                                           days.                                                     Eruption:    on those vaccinated:                                                        no. 319 (9)                                                                           in the thorax region with the                                                 8th and 9th days                                                      no. 319 (8)                                                                           in the thorax region the 7th                                                  8th and 9th days                                                      on the witnesses:                                                             no. 319 (10)                                                                          in the thorax region the 8th                                                  and 9th days.                                              ______________________________________                                    

Antibodies preventing hemagglutination after the 8th week:

    ______________________________________                                        Vaccinated         Witnesses                                                  ______________________________________                                        No. 17 1/23        No. 19 negative                                            No. 18 1/47                                                                   No. 14 1/47        No. 16 negative                                            No. 11 1/79        No. 13 negative                                            No. 12 1/39                                                                   No. 8 1/20         No. 10 negative                                            No. 9 1/47                                                                    No. 5 1/32         No. 7 negative                                             No. 6 1/20                                                                    No. 1 1/112        No. 3 negative                                             No. 2 1/56         No. 4 negative                                             ______________________________________                                    

We claim:
 1. A process for the production of an attenuated strain ofmeasles virus effective for the production of an attenuated measlesvirus vaccine, which comprises:selecting an Edmonston strain of measlesvirus which has undergone a plurality of culture passages in variousculture media, and taking a sample of the last culture passage;effecting a plurality of culture passages of said sample in primarycultures of fibroblasts of chicken embryos; inoculating a sample fromthe last culture passage and effecting a culture passage thereof in aprimary culture of fibroblasts of chicken embryos thereby obtainingclones of said virus; isolating clones of measles virus obtained in thepreceding step, and selecting those which are thermosensitive at atemperature between about 32° C. and 37° C.; among the thermosensitiveclones which have been selected, making another selection of the oneswith moderate cytopathogenic effect; and finally isolating from thelatest selection, the one which shows highest seroconversion on monkeys,thus producing an attenuated strain of measles virus.
 2. A processaccording to claim 1, wherein said Edmonston strain has undergonetwenty-four (24) culture passages in human kidney cells, twenty-eight(28) culture passages in human amniotic cells, six (6) culture passagesin the amniotic sac of embryonated eggs and thirteen (13) culturepassages in cells of chicken embryos, followed by eleven (11) culturepassages in cells of chicken embryos.
 3. A process according to claim 2,which comprises:effecting a first culture passage of said virus samplein a primary culture of fibroblasts of chicken embryos maintained at 33°C., followed by a second culture passage in a primary culture offibroblasts of chicken embryos at 33° C.
 4. A process according to claim1, wherein clones of measles virus are isolated by inoculating thestrain derived from the last culture passage in a primary culture offibroblasts of chicken embryos, in petri dishes kept in an incubator ina 5% CO₂ enriched atmosphere during about 2 hours at about 32° C., afterwhich the cultures are drained and washed with phosphate buffer salineand a culture medium is added and incubation is allowed to take placefor four to six days at 32° C., after which the liquid medium isreplaced by a methyl cellulose medium and the cultures are againincubated at 32° C. for a period of about 3 to 4 days, until plaquesappear and different clones can be separated.
 5. A process according toclaim 4, wherein forty-seven (47) samples of virus are selected from theseparated clones, and are incubated for an incubation period of about 14days, after which seven are retained which are thermosensitive at atemperature between about 32° C. and 37° C., each of the seven samplesselected is treated for the purpose of isolating clones by producingplaques, each plaque is again isolated in order to select three cloneswith moderate cytopathic effect, and finally, of the three clonesselected, the one which has highest seroconversion on monkeys isseparated and is frozen at a temperature of -75° C.
 6. A processaccording to claim 5 which comprises suspending the attenuated measlesvirus in a harvest medium and adding a stabilizer comprising dextran,monosodium glutamate, d-sorbitol and normal serum albumin (human).
 7. Aprocess according to claim 6, wherein the amount of stabilizer in thecomposition is the following:

    ______________________________________                                        dextran:              5.0%                                                    monosodium glutamate: 1.0%                                                    d-sorbitol:           1.0%                                                    normal serum albumin (human)                                                   (25% solution):      0.5%.                                                   ______________________________________                                    


8. A measles vaccine whenever produced by the process of claim 1, 5 or6.
 9. A measles vaccine whenever produced by the process of claim 7 or8.